We propose that some proteins involved in Parkinson's Disease (PD) are poorly degraded by autophagic/lysosomal pathways, resulting in disruption of normal cytosolic dopamine homeostasis, intracellular oxyradical stress, and selective degeneration of substantia nigra (SN) neurons. The central proposals are 1) reactions requiring oxidized dopamine underlie the selectivity of neuronal death in the SN, 2) aberrant autophagic degradation of alpha-synuclein dysregulates cytosolic dopamine. To test these hypotheses, we will use a range of biochemical, genetic, and physiological approaches. First, using SN cultures derived from tyrosine hydroxylase (TH) knockouts, alpha-synuclein knockouts, and their sibling wild-type animals, we will confirm if the presence of dopamine is required for selective dopaminergic neuronal death. Second, as most long-lived cytosolic proteins are degraded through lysosomal (a.k.a. autophagic) pathways, we will determine the half-lives and proteolytic pathways followed by proteins implicated in familiar PD and determine whether proteins implicated in familial PD, including alpha-synuclein are normally degraded in lysosomes. Third, as amphiphilic proteins at sufficient levels can damage intracellular membranes, we will assay effects of these proteins on membrane integrity and adapt methods to reconstruct fusion between AGs and between AGs and lysosomes. We will also use real time vital microscopy to document changes in fusion events related to changes in intracellular levels of PD mutant proteins in cultured neurons. Finally, as we propose an interaction between alpha-synuclein mutations, aberrant protein degradation, and ultimately, that a pathogenic increase in cytosolic dopamine may underlie PD we will use a new approach, intracellular patch electrochemistry (IPE), to directly measure cytosolic catecholamines under a range of conditions. In summary, this series of experiments will test the possibility that altered lysosomal degradation of alpha-synuclein and consequent alterations in cytosolic dopamine levels provides an initial, upstream cause of at least some forms of PD.